Angiogenesis, the formation of new blood vessels from existing ones, is essential to many physiological and pathological processes. Normally, angiogenesis is tightly regulated by pro- and anti-angiogenic factors, but in the case of diseases such as cancer, ocular neovascular diseases, arthritis, and psoriasis, the process can go awry. Folkman, J., Nat. Med., 1:27-31 (1995). There are a number of diseases known to be associated with deregulated or undesired angiogenesis. Such diseases include, but are not limited to, ocular neovascularisation, such as retinopathies (including diabetic retinopathy), age-related macular degeneration, psoriasis, hemangioblastoma, hemangioma, arteriosclerosis, inflammatory disease, such as a rheumatoid or rheumatic inflammatory disease, especially arthritis (including rheumatoid arthritis), or other chronic inflammatory disorders, such as chronic asthma, arterial or post-transplantational atherosclerosis, endometriosis, and neoplastic diseases, for example so-called solid tumors and liquid (or hematopoietic) tumors (such as leukemias and lymphomas). Other diseases associated with undesired angiogenesis will be apparent to those skilled in the art.
Although many signal transduction systems have been implicated in the regulation of angiogenesis, one of the best-characterized and most endothelial cell-selective systems involves the Tie-2 receptor tyrosine kinase that is selectively expressed within the vascular endothelium (referred to as “Tie-2” or “Tie-2R” (also referred to as “ORK”); murine Tie-2 is also referred to as “tek”) and its ligands, the angiopoietins (Yancopoulos, G. D., et al., Nature 407 [2000] 242-48; Gale, N. W. and Yancopoulos, G. D., Genes Dev. 13:1055-1066 [1999]).
There are 4 known angiopoietins; angiopoietin-1 (“Ang-1,” alternatively abbreviated with ANGPT1 or Ang1) through angiopoietin-4 (“Ang-4”). These angiopoietins are also referred to as “Tie-2 ligands” (Davis, S., et al., Cell, § 7:1161-1169 [1996]; Grosios, K., et al, Cytogenet Cell Genet, § 4:118-120 [1999]; Holash, J., et al, Investigative Ophthalmology & Visual Science, 42:1611-1625 [1999]; Koblizek, T. I., et al, Current Biology, S:529-532 [1998]; Lin, P., et al, Proc Natl Acad Sci USA, 95:8829-8834 [1998]; Maisonpierre, P. C, et al, Science, 277:55-60 [1997]; Papapetropoulos, A., et al, Lab Invest, 79:213-223 [1999]; Sato, T. N., et al, Nature, 375:70-74 [1998]; Shyu, K. G., et al, Circulation, 95:2081-2087 [1998]; Suri, C, et al, Cell, <37:1171-1180 [1996]; Suri, C, et al, Science, 252:468-471 [1998]; Valenzuela, D. M., et al, Proceedings of the National Academy of Sciences of the USA, 96:1904-1909 [1999]; Witzenbichler, B., et al, J Biol Chem, 273:18514-18521 [1998]).
Both Ang-1 and -2 bind to Tie-2 with an affinity of 3 nM (Kd) (Maisonpierre, P. C., et al., Science 277 (1997) 55-60). Whereas Ang-1 binding to Tie-2 stimulates receptor phosphorylation in cultured endothelial cells, Ang-2 has been observed to both agonize and antagonize Tie-2 receptor phosphorylation (Davis, S., et al, [1996], supra; Maisonpierre, P. C., et al, [1997], supra; Kim, I, J. H. Kim, et al, Oncogene 19(39): 4549-4552 (2000); Teichert-Kuliszewska, K., P. C. Maisonpierre, et al, Cardiovascular Research 49(3): 659-70 (2001)). The phenotypes of mouse Tie-2 and Ang-1 knockouts are similar and suggest that Ang-1-stimulated Tie-2 phosphorylation mediates remodeling and stabilization of developing vessels in utero through maintenance of endothelial cell-support cell adhesion (Dumont, D. J., et al, Genes & Development, 8:1897-1909 [1994]; Sato, T. N., et al, Nature, 376:10-14 [1995]; Suri, C, et al, [1996], supra). The role of Ang-1 in vessel stabilization is thought to be conserved in the adult, where it is expressed widely and constitutively (Hanahan, D., Science, 277:48-50 [1997]; Zagzag, D., et al, Experimental Neurology, 59:391-400 [1999]). In contrast, Ang-2 expression is primarily limited to sites of vascular remodeling, where it is thought to block Ang-1 function, thereby inducing a state of vascular plasticity conducive to angiogenesis (Hanahan, D., [1997], supra; Holash, J., et al, Science, 284:1994-1998 [1999]; Maisonpierre, P. C, et al, [1997], supra).
Human angiopoietin-2 (Ang-2) (alternatively abbreviated with ANGPT2 or Ang2) is described in Maisonpierre, P. C., et al., Science 277 (1997) 55-60 and Cheung, A. H., et al, Genomics 48 (1998) 389-91. Numerous published studies have purportedly demonstrated vessel-selective Ang-2 expression in disease states associated with deregulated angiogenesis (Bunone, G., et al, American Journal of Pathology, 155:1961-1916 [1999]; Etoh, T., et al, Cancer Research, 67:2145-2153 [2001]; Hangai, M., et al, Investigative Ophthalmology & Visual Science, 42:1611-1625 [2001]; Holash, J., et al, [1999] supra; Kuroda, K., et al, Journal of Investigative Dermatology, 116:113-120 [2001]; Otani, A., et al, Investigative Ophthalmology & Visual Science, 40:1912-1920 [1999]; Stratmann, A., et al, American Journal of Pathology, 153: 1459-1466 [1998]; Tanaka, S., et al, J Clin Invest, 203:34-345 [1999]; Yoshida, Y., et al, International Journal of Oncology, 25:1221-1225 [1999]; Yuan, K., et al, Journal of Periodontal Research, 35:165-171 [2000]; Zagzag, D., et al, [1999] supra). An effective anti-Ang-2 therapy will benefit a vast population of patients with angiogenesis-associated diseases, such as cancer, retinopathies, arthritis, and psoriasis.
There is a great need, therefore, to identify new compounds that specifically recognize and bind Ang-2. Such compounds would be useful for diagnostic screening and therapeutic intervention in disease states that are associated with Ang-2 activity. Accordingly, it is an object of the present disclosure to provide specific binding compounds of Ang-2 for modulating Ang-2 activity. Such compounds disclosed herein take the form of muteins derived from human lipocalin 2 (also known as neutrophil gelatinase associated lipocalin, “hNGAL”).